CD4+CD25+Foxp3+ T regulatory cells (Tregs) and dendritic cells (DCs) are central to the immunosuppressive environment characteristic of many types of cancer including breast cancer. However, the physiologic development of Tregs and DCs under such conditions is unclear. Preliminary studies suggest that monocytes, which traffic to inflamed tissues, interact with Tregs in situ and instruct monocytes to form specialized dendritic cells (DCTreg) that trigger naive T cells to differentiate into additional Tregs. The goal of this project is to understand the mechanisms governing human DCTreg formation and function such that in situ analysis of DCTreg is possible in diseases such as cancer. RESEARCH DESIGN AND METHODS;Aim 1 proposes to test the hypothesis that human DCTreg are stable and committed immunoregulatory cells that express unique surface or intracellular markers. These markers will be identified through the utilization of 12 color flow cytometry and gene expression profiling. The phenotypic and functional stability of DCTreg will be assessed by incubating immature and mature DCTreg with cytokines or bacterial stimuli that strongly induce proinflammatory responses. After treatment, DCTreg will be analyzed by flow cytometry for the expression of DC and DCTreg specific markers and assessed for their capacity to induce Treg formation from naive T cells in a mixed lymphocyte reaction (MLR). Aim 2 will elucidate the mechanism of DCTreg formation and function through the utilization of neutralizing antibodies directed against candidate markers and any DCTreg specific markers identified in Aim 1. Such antibodies will be added during monocyte-Treg cocultures and during MLRs. DCTreg formation and function will be assessed by flow cytometry and by their capacity to induce Treg formation from naive T cells during a MLR. Aim 3 will address the presence of activity of DCTreg in breast cancer through the utilization of fluorescence activated cell sorting. In these experiments, lineage-HLA-DR+DC-SIGN+ DCs will be isolated from breast cancer tissue and analyzed for their expression of DCTreg specific markers and subjected to the aforementioned functional analysis. RELEVANCE: Regulatory T cells accumulate in breast cancer tissue and actively inhibit the immune response. Our studies demonstrate that these cells induce the formation of a second cell type, which triggers the formation and multiplication of new regulatory T cells that may overwhelm the ability of the immune system to mount an effective response against the tumor. The proposed studies will provide mechanistic insights into this previously unknown pathway of immune regulation and will reveal novel therapeutic targets that may interrupt this pathway and amplify anti-tumor responses.